Electrophoresis is a well established method for the separation of biochemicals. It is especially useful in the analysis of proteins found as complex mixtures in physiological fluids and tissues. Electrophoretic systems can also be used to separate mixtures of DNA and RNA fragments in the sequencing of macromolecules.
Typically, electrophoresis is carried out in a separation medium such as a gel of agarose or polyacrylamide. These gels are either cast in molds consisting of two glass plates to form a slab or in glass tubes to form a cylindrical gel. Gels are formed in the presence of buffers to control the pH environment of the separation as well as the electrical conductivity of the gel. Simple zonal electrophoresis separates molecules under the influence of an electric field according to their electrophoretic mobility which is a complex function of the charge and size of the protein macromolecule. Isoelectric focusing is an electrophoretic method which uses media not restrictive to the passage of protein and depends on the function of a pH gradient to provide a means for separating proteins on the basis of their isoelectric points. The conventional electrophoresis process may also be modified by the addition of a detergent to the protein mixture and the separating gel to effect a coating of the protein molecule to provide a uniform charge so that separation proceeds only on the basis of molecular weight. Molecular weight separation may also be achieved by adjusting the gel composition to create a network of pore sizes which provides a sieving action to separate the molecules.
In practice, charge and size separation may be combined to increase resolution of the separation of a protein mixture. This process, 2-dimensional electrophoresis, can separate hundreds of components from physiological fluids.
In order to effect the electrophoretic separation, a means for connecting an electric field to the separating gel is needed. Usually this is done by immersing the ends of the gel slab or cylinder in reservoirs of electrically conductive buffer. These are connected by platinum or carbon electrodes immersed in the fluid to the positive and negative terminals of a power supply which establishes a voltage gradient of from 100-3000 V across the separating gel to drive the molecules in the mixture through the gel matrix. This method of attaching the gel to the power supply requires large volumes of buffer to fill the reservoirs, immersion of the gel in buffer or connection via wicks and bulky apparatus for electrophoresis. Care must be taken in making the connection because gases are released during the separation process. These gases can impair the electrical connection to the gel.
U.S. Pat. No. 3,865,712 issued Feb. 11, 1975 seeks to alleviate the problem of bulkiness and good electrode contact by placing a filter paper wick saturated with a buffer over the gel. Next an electrode is positioned on top of the filter paper. Good contact between the electrode and the filter paper is maintained by the use of a weighted member having toothlike elevations pressing the electrode against the porous material. This has the advantage of permitting easy gas escape during electrophoresis, but in the situation where the wicklike material becomes somewhat dried out, electrical continuity is compromised. Furthermore, this arrangement adds unneeded bulk to the electrophoretic system.
In another effort to solve the problem of providing adequate electrode contact, Tocci in U.S. Pat. No. 3,715,295 issued Feb. 6, 1973 describes an electrophoretic device in which wells are formed at either end of the gel and a semi-solid buffer is placed in the wells over wire electrodes or electrodes which may be painted or printed in the well. While this arrangement is a significant improvement and certainly a step in the right direction it still does not provide a compact, miniaturized package in which good electrical contact with the gel is always maintained. Furthermore, this arrangement does not lend itself to use with the 2D gel electrophoresis techniques now coming into increasing use.